Tas1r3), have a gene expression signature reminiscent of Microfold (M) cells, a central player in immune surveillance in the mucosa-associated lymphoid tissue (MALT) such as those in the Peyer’s patch and tonsils. Administration of tumor necrosis factor ligand superfamily member 11 , a growth factor required for differentiation of M cells, dramatically increased M cell proliferation and marker gene expression in the taste papillae and in cultured taste organoids from wild-type (WT) mice. Taste papillae and organoids from knockout mice lacking Spib (SpibKO), a RANKL-regulated transcription factor required for M cell development and regeneration on the other hand, failed to respond to RANKL. Taste papillae from SpibKO mice also showed reduced expression of NF-κB signaling pathway components and proinflammatory cytokines and attracted fewer immune cells. However, lipopolysaccharide-induced expression of cytokines was strongly up-regulated in SpibKO mice compared to their WT counterparts. Like M cells, taste cells from WT but not SpibKO mice readily took up fluorescently labeled microbeads, a proxy for microbial transcytosis. The proportion of taste cell subtypes are unaltered in SpibKO mice; however, they displayed increased attraction to sweet and umami taste stimuli. We propose that taste cells are involved in immune surveillance and may tune their taste responses to microbial signaling and infection.The oral microbiome is second only to its intestinal counterpart in diversity and abundance, but its effects on taste cells remains largely unexplored. Using single-cell RNASeq, we found that mouse taste cells, in particular, sweet and umami receptor cells that express taste 1 receptor member 3 ( In mammals such as the mouse, most taste buds are found in three types of taste papillae on the dorsal surface of the tongue ,2. Among16s RNA sequencing and metagenomics studies have shown that the oral cavity including the tongue dorsum is colonized by a diverse array of microbial species –11. UnliSpib, a transcription factor that orchestrates the later stages of M cell differentiation  were held constant for experiments. Colocalization counts were made using QuPath software from Advanced Cell Diagnostics. Cell boundaries were detected automatically based on DAPI staining. The fidelity of each cell boundary was confirmed by manual inspection. The number of fluorescent spots in each channel  per cell were extracted and used for colocalization counting. Data from more than 2 nonconsecutive sections from 2 mice were pooled.Immunostaining of sections containing CVP, FOP, or FFP papillae was done as described ,74. Brie2 (5 mM), EGTA (10 mM), and sucrose . After 3 × 5 min washing with 1× PBS, organoids were blocked for 45 min with SuperBlock blocking buffer (Thermo Fisher cat. no. 37515) supplemented with 0.3% (v/v) Triton X-100 and 2% (v/v) donkey serum and then incubated at 4°C overnight with the desired primary antibodies. They were washed 3× for 5 min with 1× PBS and incubated for 1 h with species-specific secondary antibodies (1:500) at room temperature. 6-Diamidino-2-phenylindole  in deionized water was used to visualize the nuclei following secondary antibody. All images were captured with the TCS SP2 spectral confocal microscope (Leica Microsystems). Scanware Software (Leica Microsystems) was used to acquire z-series stack captured at a step size of 0.5 μm. Images were scanned using a 512 × 512 pixel format, scan lines were averaged twice, and frames were scanned 3 times. Acquisition parameters  were held constant for experiments with antibodies and for controls without antibodies. Digital images were cropped and arranged using Photoshop CS (Adobe Systems). Colocalization for each taste cell marker was done using images from at least 2 sections showing entire CV papillae or foliate papillae of each mouse was counted (N = 3). Only those taste cells for which the entire cell body and nucleus could be visualized were counted.Immunostaining of taste organoids was done as previously described ,72. BrieN = 5).Mice anesthetized by an intraperitoneal injection (10 ml/kg) of a mixture of ketamine (4.28 mg/ml), xylazine (0.86 mg/ml), and acepromazine (0.14 mg/ml) were administered fluorescent polystyrene latex nanoparticles  . After 2 h, tongues were isolated and fixed using 4% paraformaldehyde in 1× PBS. Then, frozen sections of 10 μm thickness were obtained as described above and stained with an antibody specific for T1R3. Confocal images were captured as described above. Quantitative analysis of fluorescent beads uptake from lingual epithelium containing CVP was done as previously described . AnalysiSpibKO and their littermates Spib+/+ mice were tested at the same time in parallel. Each mouse was tested with all the compounds. After each session, mice were allowed to recover for 48 h with free access to food and water. Body weight of the mice was monitored daily, and the mice at or over 85% their initial were used. The ratio of taste stimulus to water licks was calculated by dividing the number of licks for taste compounds by the number of licks for water presented in the parallel test session. Lick ratios >1 indicate preference behavior to the taste compound, and lick ratios <1 indicate avoidance behavior to the taste compound.Brief access tests were conducted using the Davis MS-160 mouse gustometer  as described ,74,75. Tt tests of cell counts and qPCR data. Data from taste behavioral tests were compiled using Microsoft Excel. For statistical analyses of behavioral responses, two-way ANOVA and post hoc pairwise multiple comparisons with Tukey’s correction were used to evaluate the difference between genotype and concentration using the tidyverse and emmeans packages in R. The level of statistical significance was set at p < .05. p-Values < .05 were considered significant for paired t tests of qPCR results. All data used for statistical analyses are provided in the Supporting information table. The code used for this analysis is provided in the Supporting information file Prism (GraphPad Software) was used for statistical analyses, including calculation of mean values, standard errors, and unpaired S1 DataThe figures and the sheets in the excel file corresponding to them are as follows: (XLSX)Click here for additional data file.S1 CodeThe code for analysis of gustometer data in (DOCX)Click here for additional data file.S1 FigA) End point PCR (35 cycles) of Gapdh (housekeeping control gene) Gnat3 (taste tissue control gene), Gp2, Ccl9, Marcksl1, Anxa5, Sgne1, and Spib from cDNA prepared CVP, non-taste lingual epithelium (NT), and Peyer’s Patch (PP). Gp2, Ccl9, Sgne1, and Spib are expressed in CVP and PP, but not in NT. Marcksl1 and Anxa5 are expressed in all tissues, although at lower levels in the NT. Lines on the right indicate position of molecular weight markers. (B) qPCR analysis of above genes expression in CVP and NT confirms that all M cell marker genes are highly expressed in CVP, while they are expressed at lower levels or not at all in NT. The expression of each marker gene is plotted as the logarithm of the ratio between its cycle threshold values to those of Gapdh. Individual datapoints from each cDNA sample in shown in blue dots (the underlying data can be found in Data B in S1_Data). (C-D) RNAscope hybridization using an Spib-specific probe set produced strong signals in subsets of taste cells in CVP and FFP. (E-G) Indirect immunofluorescence confocal microscopy of cryosection from taste papillae and PP stained with an SPIB antibody shows strong nuclear staining in subpopulations of taste cells in CVP, FOP, and PP. (H-I) Absence of SPIB signal in CVP and PP of SpibKO mice proves the specificity of SPIB antibody. (J) Omission of the primary antibody (W/O) demonstrates low nonspecific background from secondary antibody in CVP. Nuclei are counter stained with DAPI in panels C-J. Scale bars, 50 μm. **p < .01, ***p < .001.((TIF)Click here for additional data file.S2 FigA-C and G-I) or the type III taste receptor marker CAR4 (D-F and J-L) in the CVP. Merged images show GP2 and CCL9 are coexpressed with TRPM5 , but not CAR4 . Nuclei are counterstained blue with DAPI. Scale bar, 50 μm.Double-labeled immunofluorescence confocal microscopy of CVP sections with antibodies against M cell markers GP2 and CCL9, with the type II taste cell marker TRPM5 ((TIF)Click here for additional data file.S3 FigA), GP2 (B), and CCL9 (C) show no staining in CVP from Pou2f3 knockout mice that lack all type II taste cells. Nuclei are counterstained blue with DAPI. Scale bar, 50 μm.Immunofluorescence images with antibodies against SPIB ((TIF)Click here for additional data file.S4 FigA-H) Indirect immunofluorescence confocal microscopy of CVP sections from WT mice treated or untreated using RANKL stained with antibodies against M cell markers GP2, SPIB, CCL9, and MARCKSL1. The result showed that administration of RANKL led to a dramatic increase in the proportion of taste cells expressing these proteins. (I-K) qPCR analysis showed that all the above marker genes and Spib were significantly up-regulated after RANKL treatment in WT but not SpibKO mice. ND, not done. (M) RANKL treatment did not affect the expression level of M cell marker genes in NT lingual epithelium. Data are means ± SEM. (The data underlying the graphs can be found in Data I-M in S2_Data.) **p < .01, ***p < .001. Nuclei are counterstained blue with DAPI. Scale bar, 50 μm.((TIF)Click here for additional data file.S5 FigA-L) Indirect immunofluorescence confocal microscopy of taste organoids from control and SpibKO mice showing kinetics of expression of the M cell markers MARCKSL1 (A-D), CCL9 (E-H), and GP2 (I-L) 0–3 days after RANKL treatment. (M-P) qPCR analysis of the expression of M cell markers in cultured taste organoids after RANKL treatment. The kinetics of expression of GP2, CCL9, and MARCKSL1 after RANKL treatment were distinct at both protein (A-L) and mRNA (M-P) levels. SpibKO mice failed to up-regulate all M cell marker genes (M-P) upon RANKL administration. (The data underlying the graphs can be found in Data M-P in S3_Data.) Data are mean means ± SEM. Scale bars, 50 μm.((TIF)Click here for additional data file.S6 FigSpibKO mice. (A-B) The expression of the transcription factors Nfkb1, Nfkb2, Rela, Relb, Rel, Irf6, and regulator proteins Tollip, Myd88, Irak3, and Irak4 belonging to the NF-κB signaling pathway and Irf6 are significantly down-regulated in SpibKO mice. (C) Compared to WT mice, SpibKO mice showed lower expression of proinflammatory cytokines Il1b, Il6, Tnf, and Mcp, but the expression of anti-inflammatory cytokines Il10 and Il12 did not change. (The data underlying the graphs can be found in Data A-C in S4_Data.) Data are mean means ± SEM. *p < .05.qPCR analysis of proinflammatory cytokines and the components of NF-κB signaling pathway in WT and (TIF)Click here for additional data file.S7 FigSpibKO mice. (The data underlying the graphs can be found in Data A-F in S5_Data.) Data are means ± SEM. *p < 0.05, **p < .01, ***p < .001.Compared to WT mice, LPS administration triggered exaggerated cytokine expression in CVP of (TIF)Click here for additional data file.S8 FigA-H) Indirect immunofluorescence confocal microscopy of CV sections from WT and SpibKO mice immunostained for type II cells using markers T1R3 , TRPM5 , GNAT3 , and type III taste cells with antibodies against CAR4 . Nuclei are counterstained with DAPI (blue). (I) Compared to WT mice, the proportion of taste receptor cells in SpibKO mice were unaltered. (The data underlying the graphs can be found in Data I in S7_Data.) (J) qPCR analysis of expression of the corresponding genes confirms this observation (Data J in S7_Data). Data are mean means ± SEM with individual data points from replicates shown as black/blue dots. Scale bar, 50 μm.((TIF)Click here for additional data file.S1 TableNormalized scRNASeq data from T1r3-GFP (Tas1-10), Gnat3-GFP (Gnat 1–10), and Gad1-GFP (Gad1-12) cells are shown. The statistical comparison shown was done between T1r3-GFP and Gad1-GFP cells using DESeq2.(XLSX)Click here for additional data file.S2 TableSpib, Gp2, Tnfrsf11a, Tas1r3, Gnat3, Trpm5, and Car4, and singly and doubly labeled cells were counted. Numerators are the numbers of taste cells expressing both gene 1 and gene 2. Denominators are the numbers of taste cells expressing gene 1. Taste cells expressing both gene 1 and gene 2 as a percentage of those expressing gene 1 are shown in parentheses. ND, not determined.RNAscope Hiplex assay was done in CVP with probe sets against (DOCX)Click here for additional data file.S3 TableTaste cells from CVP and FOP were doubly stained for SPIB and a second antibody against T1R3, GNAT3, TRPM5, or CAR4, and singly and doubly labeled cells were counted. Numerators are the numbers of taste cells expressing both gene 1 and gene 2. Denominators are the numbers of taste cells expressing gene 1. Taste cells expressing both gene 1 and gene 2 as a percentage of those expressing gene 1 are shown in parentheses. ND, not determined.(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table*RRID, Research Resource Identifier.(DOCX)Click here for additional data file.S1 Raw Image(PDF)Click here for additional data file. 22 Apr 2022Dear Dr Sukumaran, Thank you for submitting your manuscript entitled "Sweet taste receptor cells may participate in mucosal immune surveillance" for consideration as a Research Article by PLOS Biology.Your manuscript has now been evaluated by the PLOS Biology editorial staff as well as by an academic editor with relevant expertise and I am writing to let you know that we would like to send your submission out for external peer review. 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Please don't hesitate to contact us if you have any questions or comments.Sincerely,Ines--Ines Alvarez-Garcia, PhDSenior EditorPLOS Biologyialvarez-garcia@plos.org------------------------------------------------------------------Reviewers' commentsRev. 1:These authors demonstrate that taste receptor cells express key genes and proteins involved in M cell surveillance of the gut microbiome and might play a similar role in the oral cavity. This is an exciting study that opens the door to novel taste cell-microbial interactions. The work is also timely given the altered oral microbiome in COVID-19 patients and a possible link to taste loss. The authors used compelling mix of in vivo and organoid models. The manuscript is generally well-written and most of my suggestions are relatively minor. Most significant are the lengthy presentation of results in the introduction and questions about interpreting results.* In the title and throughout the manuscript the focus is on sweet taste receptor cells. While that might be shorthand for sweet- and umami-sensing type II cells I don't find "STRC" accurate given expression and behavioral data.* The introduction would be improved by summarizing the main findings more concisely.* Methods1) Are mice housed in a SPF barrier facility which relates to microbial exposure?2) What is the route and dose of LPS administration?3) Data were analyzed by two-way ANOVAs followed by t tests as multiple comparisons. Were P values adjusted to control Type I error introduced by multiple t tests?4) Provide details for confocal imaging including objective, zoom and optical thickness of z scans.* Figure 1L. What are the heavily stained SPIB+ cells at the base of foliate papillae? If they are glands this might be discussed later in comparison to the gut.* Figure 2. The two lower magnification panels make it difficult to see co-localization vs. background.* Page 8 line 181. The statement that "Collectively, the results above conclusively show that STRCs express several M cell marker genes" in not precise. Some of the genes were co-localized with TRPM5/type II markers rather than T1R3.* Figure S2 and accompanying text/legend. The results demonstrate an increase in M cell marker genes upon treatment with RANKL but not proliferation, which was not directly tested.* Legends for Fig. S3 and 5 have the same title. It was also not clear why some of the results were presented as supplementary figures rather than included in the main text unless there are journal limits.* Figure 5. The images showing bead uptake are not very convincing. Were beads at the apical surface of the taste counted? It appears that only 2-3 beads are present within the taste bud.* To my knowledge, M cells fail to develop from progenitors in SPIB KO mice. In contrast there were no differences in the different types of taste cells in KO mice. The difference in the gut and taste cell phenotypes in SPIB KO mice is striking and could be added to the discussion section comparing and contrasting taste cells and M cells.Rev. 2:This manuscript reports that the subset of taste receptor cells that respond to sweet tastes express markers for M cells of the immune system, including SPIB. A comparison between the taste cells and immune cells is further supported by experiments showing that administration of RANKL leads to an increase in the number of cells that express these markers, while expression of these markers is lower in a Spibko mouse strain. Most remarkably, the authors find that Spibko mice show a heightened preference for sucrose and MSG over water, as compared to WT mice. Overall, there are some interesting and potentially very important findings in this manuscript.Major:1. Much of the paper rests on immunocytochemistry to define the population of cells that express SPIB. However, because SPIB is nuclear, and the taste cell markers are on the plasma membrane (or cytosol), it is sometimes difficult to tell in which cells they are co-expressed (and how this was assessed by the authors). This is made more difficult by quality of some of the images. One solution would be to use the TRPM5-GFP mouse strain generated by this PI, which should show better colocalization. It would also be helpful if the authors added a Venn diagram to show cell counts .2. Orgenoid IHC is not convincing given low resolution images and poor morphology of the cells, and adds little to the manuscript. It could be put in a supplementary figure.3. Images showing bead uptake are not convincing - the beads appear to be stuck at what appears to be the apical surface of the tastebud (labeling the taste pore would be helpful). The negative control does not have a similar number of T1R3 labelled cells. Did the investigators ever observe beads inside the cells? This should be shown if possible.4. The increased preference for sucrose and MPG is impressive. But some critical questions need to be answered. First, it is surprising that a similar change in preference is not seen for sucralose. How can this be explained? And without a mechanism, this change in behavior could be due to global effects of the knockout . To show that there is a change in the peripheral response of taste cells, and connect the results to the main findings of the papers, the authors should, at a minimum, show results from gustatory nerve recording.5. Is there something about this strain or the way that the animals were tested that makes them so insensitive to ionic tastes? First, the mice continue to lick high concentrations of NaCl (300 mM and 600 mM) that are typically very aversive . Also, concentrations of citric acid that are typically strongly aversive (30 mM - 300 mM) appear to not be aversive or only mildly aversive, and the concentrations in the figure are different from those in the methods.Minor:Fig 1 A - please include MW markersFig 1B - the gene "Spi-B" is presumably Spib?Fig 1C-J - label figure with name of probe Fig K-N - In the absence of taste cell markers, itt is hard to tell where the taste buds are in the IHC, making the expression of SPIB not very convincing. Same for KO. I suggest leaving this out, and simply showing the IHC in Fig 2, along with the KO controls (counterstained for taste cell markers).Fig 6 A - SpiB knockout - should be Spib? (gene name)Methods: Was tissue fixed in PFA for 1h or overnight (line 452)?Rev. 3:In this manuscript the authors suggest that a subset of sweet sensitive taste receptor cells in mouse taste buds functions in immune surveillance of pathogens in the oral cavity. The mechanistic hypothesis is very exciting and very timely given the impact of SARS-CoV2 on smell and taste function. They interrogate their published scRNAseq dataset of 3 different taste bud cell types for genes expressed by microfold (M) cells that function in other immune tissues and show that several of these are expressed in taste cells. They first use immunostaining to show several microfold cell marker proteins, including Spib, Gp2, and Ccl9, are co-expressed in taste bud cells. They then use organoid technology to activate the immune response with the RANKL, the ligand for Tnfrsf11a, which they find enriched in taste bud cells in the RNAseq dataset, and show substantial upregulation of several M cell gene products in response to RANKL. They further show that Spib is required for this response in organoids and in mice. In vivo, M cells have epithelial transcytosis function and can take up fluorescent beads, and the authors show data that Tas1r3+ cells in mouse taste buds also take up these beads and uptake is Spib-dependent. Finally, they use a behavioral assay to show that compared to controls, Spib-cKO mice have altered taste responses to sweet and umami tastants, but not to bitter, salt or sour, and suggest that in addition to functioning in immune surveillance, sweet taste cells might "might modulate taste preference in response to microbial colonization and infection "While these are intriguing findings, their data do not appear to fully support a model where sweet taste receptor cells uniquely function to mediate oral immune responses. In fact there data suggest that there are complex expression patterns of M cell genes in taste epithelium and complex responses to immune challenge in taste epithelium that are not consistent with their interpretation.The authors rely on their published scRNAseq dataset of mouse CVP taste cells, made up of only 32 cells and roughly 10 cells per cell type . In table S1, the authors present M cell genes enriched in their dataset. Combined with the highly variable expression of each gene within each group, comparisons of differentially regulated genes across groups should be more cautiously interpreted by the authors here. Comparing Tas1r3+ sweet and umami type II cells vs Gad1+ type III cells for example show differences between these 2 populations, however, the authors are making the strong statement that these immune related genes are upregulated specifically in sweet taste receptor cells. Of interest, some genes they suggest are functionally involved in immune response of taste buds are actually most highly expressed in Gad1+ cells, i.e., Gp2, Tnfrsf11a. They include scRNAseq expression data also for the Gnat3+ bitter type II cells. A quick comparison of Gnat3+ and Tas1r3+ cells shows that many of these genes are not upregulated specifically in sweet cells but instead are expressed in both type II populations, e.g. Spib, or more highly, although quite variably, expressed in bitter cells e.g., Scg5, Gp2. PCR and immunofluorescence analysis  also suggests that these M cell genes are expressed in a complex pattern among bitter and sweet/umami cells, as well as in the non-taste epithelium of the tongue . The authors appear to gloss over this complexity:"Using single cell RNASeq of GFP-labeled mouse taste cells, we found that sweet taste receptor cells (STRCs) and a few other taste cell types express several M cell marker genes" It is also important to note that Tas1r3 is also expressed by umami-sensitive taste buds cells, and thus limiting the interpretation of the function of Tas1r3+ cells to sweet taste cells is an oversimplification as well.Additional experiments that would improve the manuscript would be to show explicit co-expression of M cell genes with Tas1r2 mRNA in taste tissue.Specific critiquesImmunofluorescence for GP2 or CCL9 in Fig 3 appear to show many cells in taste buds express these proteins, not just TRPM5+ taste cells, which is expressed by bitter, sweet and umami type II cells. In panel 3D-F, GP2 appears to also be expressed in a CAR4+ cell, and the extent to which GP2 is co-expressed in 3A-C is difficult to determine as immunofluorescence is brightest at the taste pore. Given the apparent high background of GP2 and CCL9 the authors should show control tissue with primary antisera omitted, especially since GP2 is a mouse antibody.Further in Fig S2, GP2 appears not to be expressed in control taste tissue, only in that from mice treated with RANKL, and with RANKL treatment many more cells appear immunopositive for GP2, CCL9 and MARCKSL1, and SPIB than in controls and that can be accounted for by expression specific to only Tas1r3+ sweet/umami cells. The authors should assess these expression patterns with TAS1R3 double labeling.In Figs 2, 3 and 5 the authors do not indicate if these are projections of confocal z-stacks or optical sections; the latter are required to determine if a single taste cell is double labeled, or if a single cell has taken up a fluorescent bead.In figure 5, how many taste buds and cells were analyzed for bead transcytosis?The authors should show individual datapoints for plots in Fig 1B, 4M-P, 5I, and 6A-F, S2I-L, S3A-C, S4A-F, S5B-F, .In table S2, how many mice were used in this analysis? 14 Sep 2022AttachmentResponse to reviewers comments.docxSubmitted filename: Click here for additional data file. 2 Nov 2022Dear Dr Sukumaran,Thank you for your patience while we considered your revised manuscript entitled "Type II taste cells may participate in mucosal immune surveillance" for publication as a Short Report at PLOS Biology. 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We will require these files before a manuscript can be accepted so please prepare and upload them now. Please carefully read our guidelines for how to prepare and upload this data: ------------------------------------------------------------------------Reviewers' comments:Rev. 3:The authors have addressed my concerns.2 minor corrections noted:Line 237 change "proliferation of M cells" to "increased expression of M cell marker genes"Line 488 methods. How long after LPS treatment were mice harvested? 29 Nov 2022Dear Sunil,We are almost ready to accept for publication your manuscript entitled "Type II taste cells participate in mucosal immune surveillance" at PLOS Biology as a Short Report. However, we have realised your manuscript currently doesn't comply with our Short Reports format as it currently has eight main figures instead of four. 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Immunodiagnostics have been widely used in the detection of disease biomarkers. The conventional immunological tests in central laboratories require expensive equipment and, for non-specialists, the tests are technically demanding and time-consuming, which has prevented their use by the public. Thus, point-of-care tests (POCT), such as lateral flow immunoassays, are being, or have been, developed as more convenient and low-cost methods for immunodiagnostics. However, the sensitivity of such tests is often a concern. Here, a fluorescence-linked immunosorbent assay (FLISA) using organic light-emitting diodes (OLEDs) as excitation light sources was investigated as a way forward for the development of compact and sensitive POCTs. Phycoerythrin (PE) was selected as the fluorescent dye, and OLEDs were designed with different emission spectra. The leakage light of different OLEDs for exciting PE was then investigated to reduce the background noise and improve the sensitivity of the system. Finally, as proof-of-principle that OLED-based technology can be successfully further developed for POCT, antibodies to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in human serum was detected by OLED−FLISA. Immunodiagnostics have become a widely used method for diagnosis of diseases and monitoring their transmission ,2. MedicThe basic components of fluorescence sensing involve a light source, filter sets and a detector. The target analytes are labelled with dyes and then excited by the light, and the detector collects the fluorescence from the dyes to compare with the reference sample. It is important that the components for POCT need to be compact enough to match the size of the system. Hence, cumbersome optical components such as lasers, filtered lamps, photomultipliers, and spectrometers are not suitable for POCT. Compact light sources such as inorganic light-emitting diodes (LEDs) and OLEDs are considered suitable candidates for POCT. Several works using compact systems based on LEDs and OLEDs to detect disease biomarkers have been reported. LEDs were used in microfluidic devices for detecting myoglobin and CK-MB . OLED-reAlthough OLEDs have attractive features for POCT, many aspects of OLEDs need to be tailored to fit the requirements of specific sensing systems and analytes. One of the challenges is the broad emission spectrum of OLEDs that causes the leakage of excitation light after filtering. Hence, rejecting the excitation light effectively and minimizing the noise of the sensing system can be crucial to achieve sensing with high sensitivity. Moreover, testing the actual disease biomarkers from the patients can be more realistic in reflecting the capability of OLEDs for POCT. Here, we demonstrate the sensing of antibodies to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from patient serum using an OLED-based system as a proof-of-principle test for sensing disease biomarkers. Sensing SARS-CoV-2 can be a good demonstration as there are various types of tests available on the market such as nucleic acid tests and serological tests. In view of the limited testing capacity caused by the rapid growth of the pandemic, it is desirable to have a sensitive, accurate, compact and convenient test for point-of-care use . The tesWe describe experimental methods and details  in this section. As this article substantially concerns instrument development, we consider improving OLED design and system design, together with test measurements in the results section.The photoluminescence excitation (PLE) and photoluminescence (PL) of phycoerythrin (PE) solutions were measured in a cuvette by a fluorimeter . The PE was prepared at 100 nM in phosphate-buffered saline (PBS). For the PLE measurement, the detection wavelength was 680 nm. For the PL measurement, the excitation wavelength was 350 nm.−7 mbar. The size of the OLEDs used in this work was 4 mm by 4 mm. For the rate of deposition, the Al anode was deposited at 3 Å/s; hole transport layer (HTL) was deposited at 0.6 Å/s; emission layer (EML), hole-blocking layer (HBL) and electron-blocking layer (EBL) were at 0.3 Å/s; electron transport layer (ETL), Ag cathode and capping layer were at 1 Å/s; The OLEDs were unloaded directly into a glove box with a nitrogen atmosphere and encapsulated with glass lids and epoxy glue.OLEDs were fabricated by thermal evaporation  at a base pressure of 3 × 10The electroluminescence (EL) spectra of the OLEDs were taken by a spectrograph  connected to a charge-coupled device (CCD) camera . For OLED characterization, a source meter  was used to scan OLED devices with constant voltage. The optical performance was measured with a multimeter  and a calibrated Si photodiode.For OLEDs driven under the pulse operation for fluorescence sensing, a pulse generator  was used to supply pulsed voltage with a width of 300 µs at 10 Hz. The fluorescence was detected by a silicon photodiode  and read out by an oscilloscope . The gain of the Si photodiode was set to be 70 dB. A 20.5 Ω resistor was connected in series for protection and to measure the current in the circuit. The voltage produced by the pulse generator was 100 V, which was distributed across OLED and resistor, resulting in a different voltage directly applied to the OLEDs due to the difference in the device structure.The basic operation of the fluorescence sensing system is to compare the difference in fluorescence intensity between samples. The fluorescence of the reference samples was first measured under the excitation of OLEDs and collected by the photodiode, then the samples with target analytes were introduced and measured using the same system setup. By comparing the difference in fluorescence signal between the target sample and reference sample, the presence of the target analytes can be determined. The result obtained by oscilloscope was processed by a computer with a Python program. The program calculated the subtracted fluorescence signal by taking the difference between the fluorescence of target sample and reference sample, and then averaged for 800 data points. The testing time for each sample in this fluorescence sensing system is less than 30 s, which is fast enough to be used in POCT.w/v Bovine Serum Albumin (BSA)  and 0.1% v/v Tween 20  at a volume of 1 mL/well for 1 h at room temperature. Slides were then incubated with a lysate of cells expressing V5-tagged SARS-CoV-2 Nucleoprotein in PBS containing 0.1% v/v Tween 20 (PBS-T), with gentle agitation, for 2 h at 4 °C. Unbound antigen was washed away with PBS-T, and the slides were blocked with PBS-T containing 5% w/v BSA and 10 µg/mL mouse anti-V5-Tag antibody containing PBS-TBV for 1 h at room temperature. Then, the blocking buffer was removed, and the slides were incubated with samples of sera from control and SARS-CoV-2-convalescent patients in a dilution of 1:80 or 1:160 in PBS-TBV, 0.5 mL/well, for 1 h at room temperature with gentle agitation. Again, the unbound antibody was washed away with PBS-T. The slides were incubated with phycoerythrin-conjugated donkey-anti-Human IgG antibody  diluted 1:1000 in PBS-TBV, 0.5 mL/well for 1 h at room temperature with gentle agitation. The unbound antibody was washed away with PBS-T and well chambers were removed. Finally, the coverslips were mounted with Citifluor AF-1.The chambered slides  were coated with mouse anti-V5-Tag antibody , 10 µg/mL in PBS, with gentle agitation overnight at 4 °C. Then, the unbound antibody was removed, and the slides were blocked with PBS containing 5% It is important to choose a suitable dye as the indicator to detect antibodies with fluorescence sensing. As OLEDs generally show broad emission spectra, reducing the long emission tail for lower background noise in fluorescence sensing is necessary . The bacN)phenyl-C](acetylacetonato)iridium(III) Ir(ppy)2(acac) doped in a co-host system formed by tris(4-carbazoyl-9-ylphenyl)amine (TCTA) and 2,4,6-tris(biphenyl-3-yl)-1,3,5-triazine (T2T) for higher conductivity and device efficiency. The overall structure of Ir−OLED is Al (80 nm)/2,2′,7,7′-tetraamino-9,9-spirobifluorene (Spiro-TTB): 2,2-dimalononitrile (F6-TCNNQ) (4 wt%) (40 nm)/1,1-bis[(di-4-tolylamino)phenyl]cyclohexane (TAPC) (10 nm)/TCTA: T2T: Ir(ppy)2(acac) (0.46: 0.46: 0.08 in wt%) (20 nm)/T2T (10 nm)/4,7-diphenyl-1,10-phenanthroline (BPhen): Cs2CO3 (40 nm)/Ag (20 nm)/N,N′-di(1-naphthyl)-N,N′-diphenyl--4,4′-diamine (NPB) (70 nm).OLEDs with green or blue emissions were designed to excite the PE. A top-emitting structure was used for OLEDs to achieve narrower emission spectra and wavelength-tuning using the microcavity effect. Furthermore, doped charge transport layers were used for making p-i-n OLEDs with high conductivity and high brightness. PE can have an efficient excitation at green wavelengths but a smaller separation between the excitation and fluorescence compared to excitation at blue wavelengths. Here, a green OLED and two blue OLEDs based on different emitters were developed to deliver different excitation spectra. The green OLED, Ir−OLED, with a similar structure to our previous work was developed for exciting PE b 18]. T. T18]. TMoreover, PE can also be excited with blue OLEDs. One blue OLED design used an efficient and stable device structure with an EML of 2,5,8,11-Tetra-tert-butylperylene (TBPe) doped in 2-methyl-9,10-bisanthracene (MADN), noted as TBPe−OLED. The structure of TBPe−OLED was Ag (100 nm)/Spiro-TTB: F6-TCNNQ (4 wt%) (170 nm)/NPB (10 nm)/MADN: TBPe (1.5 wt%) (20 nm)/Bis(8-hydroxy-2-methylquinoline)-(4-phenylphenoxy)aluminum (BAlq) (10 nm)/BPhen: Cs (55 nm)/Ag (20 nm)/NPB (40 nm). The other blue OLED design used a multi-resonance blue emitter, DABNA-2, with a narrow emission . The str2(acac), TBPe and DANBA-2 emitters have very different electroluminescence (EL) spectra. The Ir−OLED has a green emission peak at 519 nm with a full width at half maximum (FWHM) of 48 nm. The TBPe−OLED has a broad blue emission with a dominant peak at 495 nm and a secondary peak at 465 nm, and FWHM of 57 nm. The DABNA−OLED showed an emission peak at 465 nm with a FWHM of 21 nm, which is less than half of that of Ir−OLED and TBPe−OLED. The current density-voltage-luminance (J−V−L) characteristics of Ir−OLED, TBPe−OLED and DABNA−OLED are shown in 2 with a brightness of around 113,000 cd/m2. The TBPe−OLED showed a current density of 213 mA/cm2 at 5 V with a brightness of 3663 cd/m2. The DABNA−OLED achieved 496 mA/cm2 at 5 V with a brightness of around 5100 cd/m2. The higher brightness of the Ir−OLED arises from the high efficiency of the phosphorescent emitter. As shown in As shown in The fluorescence sensing system was first built to evaluate the excitation of PE using different OLEDs a. The maIn fluorescence sensing, different filter sets are needed to separate the excitation light and the sample fluorescence. SNR) was used to compare the fluorescence signal level and the background noise level. It was defined as S is the raw fluorescence signal, B is the background signal, and S−BSD is the standard deviation of the fluorescence signal with background subtracted  taken from a non-infected person (control) and coronavirus disease (COVID-19) infected person (patient) were prepared. The SARS-CoV-N was either expressed (+COVID N) or non-expressed (−COVID N), where only the expressed SARS-CoV-N can bind with the antibody. The FLISA was done with the stated procedures, and the fluorescence intensity was viewed under the fluorescence microscope . As a result, no fluorescence was observed in the control group by comparing the brightness of the photos taken from the fluorescence microscope b. The abSNR was calculated here by taking the difference in fluorescence between the signal  and reference background . If the SNRs in the three reference backgrounds are all larger than 3, the antibodies to SARS-CoV-2 are considered detectable. The results of OLED−FLISA under the TBPe−OLED excitation are shown in SNRs were lower than 3, which means no antibodies were detected under the TBPe−OLED excitation. However, for the 1:80 dilution samples excited by the DABNA−OLED, higher fluorescence intensity can be seen from the group with patient serum and expressed SARS-CoV-N . The fluorescence intensity in RS-CoV-N a. The SNr than 3 b, which r than 3 c. Additir than 3 d. TherefIt is desirable to know how OLED−FLISA compares to the commercial lateral flow antibody test. However, due to the difference in geometry and reagent, it is difficult to compare directly. Here, a rough comparison was made between our OLED−FLISA and a conventional antibody test. The same human serum samples used for OLED−FLISA without further dilution were tested in a commercial lateral flow antibody test kit (Panodyne COVID-19 antibody IgM/IgG rapid test kit). The results show that for the control test with non-infected human serum, only the control line was present . For the2. However, the actual sensing area on the slide illuminated by OLED was only 25 mm2. The geometry can be further improved by adopting the microfluidic systems used in other works for less reagent consumption and more convenient washing steps of FLISA [Since there are other OLED-based systems for sensing disease biomarkers reported, it is necessary to compare our system to the reported works ,10,11,25of FLISA ,9,25. ThIn this work, a fluorescence-linked immunosorbent assay with OLEDs as excitation light source was demonstrated for detecting disease biomarkers. Phycoerythrin was used as the labelling dye due to its advantage of broad absorption and high quantum yield. OLEDs with different emitters were developed to investigate the influence of the spectra and leakage light on the sensing system. Blue OLEDs based on TBPe and DABNA-2 were found to be most suitable for PE excitation. Finally, a fluorescence-linked immunosorbent assay was used to sense antibodies to SARS-CoV-2 in human serum. The results show successful sensing of the antibodies using the OLED with DABNA-2 as the emitter. We performed a comparison which shows that the OLED−FLISA was able to detect antibodies at much lower concentrations than the commercial lateral flow antibody test. However, we did use a larger sample volume, something that could be improved in the future using microfluidics . Althoug


Per- and poly-fluorinated alkyl substances (PFAS) are environment-persitent emerging endocrine disrupting chemicals raising health concerns worldwide. Exposure to PFAS has been associated with the imbalance of thyroid hormones. However, available studies addressing the cell mechanism underlying thyroid disrupting feature of legacy PFAS, such as perfluoro-octanoic acid (PFOA), perfluoro-octane-sulfonic acid (PFOS), and the new generation substitutes, such as C6O4, are still lacking. In this study the potential disrupting effect of PFOA, PFOS, and C6O4 on a murine thyroid cell model was assessed.A rat FRTL-5 cell line was used as the normal thyroid follicular cell model. Cell iodide-uptake, induced by thyroid stimulating hormone (TSH), was used to assess the functional impact of PFAS exposure on cell function. Tetrazolium salt-based cell viability assay and merocyanine 540-based cell staining were used to address the possible involvement of cell toxicity and membrane biophysical properties on altered cell function. The possible direct interaction of PFAS with TSH-receptor (TSH-R) was investigated by computer-based molecular docking and analysis of molecular dynamics. Evaluation of intracellular cAMP levels and gene expression analysis were used to validate the direct impairment of TSH-R-mediated downstream events upon PFAS exposure.Different from PFOS or C6O4, exposure to PFOA at a concentration ≥ 10 ng/mL was associated with significant impairment of the iodide uptake upon TSH stimulation . No impairment of cell viability or membrane stability was observed. Computational analysis showed a possible direct differential interaction of C6O4, PFOA, and PFOS on a same binding site of the extracellular domain of TSH-R. Finally, exposure to PFOA was associated with a significant reduction of downstream intracellular cAMP levels and both sodium-iodide transporter and thyroperoxidase gene expression upon TSH-R stimulation.Our data suggest that legacy and new generation PFAS can differentially influence TSH dependent signaling pathways through the direct interaction with TSH-R. In the last decades, great attention has been paid to the harmful effects of various chemicals on the environment and human health and how they interfere with hormonal function, collectively known as endocrine-disrupting chemicals (EDCs). Among EDCs, per- and poly-fluorinated alkyl substances (PFAS) are emerging chemicals raising health concerns worldwide . PFAS arThyroid hormones play crucial roles in normal neurodevelopment of the fetus and child. Many chemicals can affect the control and homeostasis of thyroid hormones, eventually leading to various adverse health effects such as neurodevelopmental disorders. PFASs have been considered thyroid disrupting chemicals since the exposure to several PFASs was significantly associated with alteration of thyroid hormone (TH) balancing , 5. Thyrin vitro experimental studies seem to confirm the detrimental effects of PFAS on thyroid cells -1,3-dioxolan-4-yl) oxy]-ammonium salt (1:1), known as C6O4, are still lacking. At present, only one study from Coperchini et al. showed no alteration of cell viability, ROS production, and cell proliferation in human thyroid cell lines exposed to C6O4, although specific endocrine-disrupting targets of thyroid cell function have not yet been evaluated . To thisMerocyanine 540 , thiazolyl blue tetrazolium bromide , nutrient mixture F-12 Ham (cat# F6636), hydrocortisone solution (cat# H6909), - bovine apo-transferrin (cat# T1428), somatostatin (cat# S1763), Gly-His-Lys acetate salt (cat# G7387), and insulin from bovine pancreas (cat# I6634) were all purchased by Merck Life Science S.r.l. . Native human thyroid stimulating hormones , were purchased by Creative BioMart Inc. . The Cyclic AMP Complete ELISA Kit (cat# ab133051), used for intracellular cAMP quantification, was purchased by Abcam . The non-radioactive iodide Assay Kit (cat# 25659), used for intracellular iodide quantification, was purchased by Cayman Chemical . Perfluoro-octanoic acid and perfluoro-octane-sulfonic acid were purchased by Wellington Laboratories . C6O4 was a kind gift from Solvay Industrial – HSE .2 and 37°C in Coon′s modified Ham′s F12 medium with 2mM Glutamine, 10μg/ml Insulin, 10nM Hydrocortisone, 5μg/ml Transferrin, 10ng/ml gly-his-lys acetate, 10ng/ml somatostatin, 5% Foetal Bovine Serum (FBS), and 1 mU/ml TSH as previously described . Cells were maintained under sterile conditions at 5% COescribed . Sub-conAccumulation of PFAS levels in FRTL-5 cell culture were measured through reversed-phase (RP) liquid chromatography coupled with triple quadrupole mass spectrometry (LC-MS/MS) as previously described 3 cells/well were seeded in 96-well microplates in 100 μL culture medium. After 24 hours of culture, cells were exposed to PFAS diluted in complete medium at concentration ranging from 0.1 to 100 ng/mL as indicated below. Triplicate cultures were established for each treatment. After 24 hours of exposure, cells were incubated for 5 hours with MTT at the final concentration of 0.5 mg/mL and then lysed with a solution of 1% sodium dodecylsulfate (SDS) in HCl 0.01 M. After an overnight treatment, the absorbance of each well at 570 nm was measured by a Bio-Rad 680 microplate reader and normalized on controls lacking PFAS exposure.The possible inhibition of cell growth by PFAS was evaluated by MTT assay as previously described . BrieflyCell membrane fluidity was assessed by the bilayer sensitive probe Merocyanin 540 (MC540), as previously described –31. BrieThe possible docking of PFAS to TSH receptor (TSH-R) was investigated through a computational approach. The structure of the extracellular domain of the human (TSH-R) is available as deposited 3D structure in the Protein Data Bank (PDB code 2XWT). The computational docking analyses were carried out by considering only this domain. Once extracted from crystallography template, TSH-R was subjected to energy minimization by Yasara Energy Minimization Server (YASARA Energy Minimization Server) to obtain an estimate of its unliganded configuration. As there are no experimental models of TSH-R in complex with TSH, and it was estimated using the experimental models of the 2 TSH subunits and taking the FSH-FSHR complex as a template, whose structure was experimentally obtained (PDB code: 1XWD).https://www.cgl.ucsf.edu/chimera/) molecular modeling software , was used to evaluate the conformations the receptor can acquire after binding with PFAS and to compare them with those of the free receptor.The molecular dynamics procedure, based on the method by Kuriata et al.  and avai® TURBO DNA-free™ Kit  according to the manufacturer’s instruction. RNA purity and concentration was assessed by NanoDrop ND-1000 .Total RNA was extracted from FRLT5 cell line stimulated with increasing concentrations of PFOA, PFOS, C6O4 using the RNeasy Mini Kit . Dnase treatment was performed using AmbioncDNA was synthesized from 500 ng of total RNA using SuperScript III  and random hexamers. Real Time PCR were performed in a 20 µl final volume containing 20 ng of cDNA, 1X Power SYBR Green PCR Master Mix , and a mix of forward and reverse primers (1 mmol/l each). The following primers were used: Nis forward 5′-TCCTCACAGGCCGTATCTCA-3′ and reverse 5′-GAAGGAACCCTGGAGGACAC-3′, Tpo forward 5′-GCATGTATCATTGGGAAGCA-3′ and reverse 5′-CGGTGTTGTCACAGATGACC-3′, Tshr forward 5′- TCATTGCCTCTGTAGACCTG-3′ and reverse 5′- TGATAACTCACTGGCGAAA-3′. Mouse Beta actin was used as a reference gene: forward 5′- GGCACCACACTTTCTACAATG-3′ and reverse 5′- TGGCTGGGGTGTTGAAGGT-3′. StepOne plus  thermocycler was used for Real Time PCR and relative quantification was performed using Delta Delta Ct (2−ΔΔCt) method.TSH-induced cAMP production in FRTL-5 cells was assessed as previously described . Upon thNon radioactive cell iodide uptake was assessed in FRTL-5 cells as previously described . BrieflyAll experiments were performed in triplicate. Data were normalized on control conditions free from PFAS exposure and TSH stimulation.Statistical analysis of the data was conducted with SPSS 21.0 for Windows . The comparison between the two groups of data, obtained from western blot analysis and cytochemical staining, were determined by paired two-tailed Student’s t-test after acceptance of normal distribution of the data with the Kolmogorov–Smirnov test. One-way ANOVA with Bonferroni correction was used for the comparison of more than 2 groups of data. Values of p<0.05 were considered as statistically significant.In order to evaluate whether the exposure to PFAS was associated with altered thyrocyte cell function, the impact on TSH-dependent iodide uptake was firstly evaluated. To this end, starved FRTL-5 were exposed for 24 hours to C6O4, PFOA, or PFOS at a concentration ranging from 0.1 ng/mL to 100 ng/mL and then stimulated with human TSH 1 mU/ml .At basal conditions, in absence of TSH stimulation, compared to the unexposed control (100.0 ± 19.0%), cell iodide uptake was significantly reduced upon NaSCN treatment . In addition, differentl from C6O4 and PFOS for which no major effects were observed, a significant reduction of basal iodide uptake was observed in cells exposed to PFOA at the concentration of 0.1 ng/mL (p= 0.0366) and at a concentration equal or greater than 10 ng/mL .F=18.56 and p<0.001, for cells exposed to PFOA at any concentration F=36.15 and p<0.001 and for cells exposed to PFOS at any concentration F=30.94 and p<0.001]. However, a peculiar trend emerged from the comparison with the CTRL condition stimulated with TSH. As expected, the treatment with NaSCN significantly reduced cell iodide uptake (p=0.045). Interestingly, no significant alteration of cell iodide uptake upon TSH stimulation was observed for cells exposed to C6O4 or PFOS , while exposure to PFOA at a concentration equal or greater than 10 ng/mL was associated with a significant reduction of this parameter , suggesting a major impact of this perfluorinated compound on TSH-mediated function in the thyrocyte model.Stimulation with TSH was associated with a significant increase of cell iodide uptake in all tested conditions [respectively, versus corresponding basal: CTRL+TSH 188.9 ± 7.9%, p= 0.0025, NaSCN+TSH 107.4 ± 29.0% p=0.045; for cells exposed to C6O4 at any concentration In order to rule out a major involvement of cell viability in the observed altered function of thryrocyte upon exposure to PFOA, an MTT-based cytotoxicity test was performed  activation and, in turn, increases intracellular levels of cyclic adenosine monophosphate (cAMP) to promote the pathways transduced by protein kinase A (PKA) . On thisThe direct phosphorylation of the cAMP-responsive element binding protein (CREB) represents a key event in TSH-receptor-related signaling, driving the expression of the downstream genes such as NIS and thyroperoxidase (TPO) , 43. Accin vitro adverse effects of legacy and new-generation PFAS on a cell model of thyrocyte function and TSH-R mediated pathways. In particular, we focused on the most peculiar markers of thyrocyte function, such as iodide uptake, for which a disrupting effect was observed, essentially for PFOA. Importantly, this effect is not due to non-specific endpoints such as cell viability or membrane biophysical properties in the range of concentrations from 0.1 to 100 ng/mL. The lack of a cytotoxic effect on thyroid cells is in agreement with a recent study on the same molecules from Coperchini et al. (in vitro results are from rat cells. However, the consistency between rat and human TSH-R can be assumed on the basis of the responsiveness of the murine model to human TSH.This is the first study to compare the i et al. . Conversi et al. , sperm (i et al. . or plati et al. . The difi et al. . The faci et al. . Interesi et al. . Accordiin vitro studies show that legacy PFAS compounds exert a cytotoxic effect on the thyroid only at very high concentrations (Results from previous studies on humans reported that significant concentrations of PFOA and PFOS can be detected in thyroid gland surgical specimens  and suggtrations . In a cotrations , 49, 50,trations , showingA remaining uncertainty of the study is related to the concentrations of C6O4 which were tested in this study, being in the same dose range reported for blood serum levels of PFOA and PFOS in population exposure studies. This is clearly a provisional setting in the absence of serum levels of C6O4 in general public or industry workers. Also, we tested these compounds independently but in real life context, a cocktail effect in which humans are exposed to a mixture of PFAS and also other endocrine disruptors is much more realistic. To this regard, a very recent study has evaluated a mixture-centered risk assessment strategy, integrating epidemiological and experimental evidence. The mixture tested included also PFAS and confirmed a detrimental effect on thyroid-related pathways . FinallyOur data suggest that legacy and new generation PFAS can influence TSH dependent signaling pathways through a possible differential direct interaction with TSH-R, according to the structural moiety of the molecule. Further study in human cell and in animal models are requested to address the consistency and reliability of these preliminary dataThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The study involved the use of a commercially available rat cell model, requiring no approval by the local Ethics Committeein vitro experiments. MSR performed gene expression analysis. DG performed computer-based molecular docking and molecular dynamics analysis. SD’A performed LC-MS analysis. AG, AF, and CF contributed to the discussion, conducted the research, and collected the data. LDT, ADN, and CF designed the study and revised the manuscript. All authors read and approved the final manuscript.LDT and ADN wrote the first draft of the manuscript, conducted the data analysis and interpretation and contributed to the collection and standardization of the data. FP performed The study was funded by a grant from Solvay Specialty Polymers Italy S.p.A. The funder had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data, or approval of the manuscript; and decision to submit the manuscript for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


To investigate the expression intensity of carbonic anhydrase IX (CA-IX) in bladder urothelial carcinoma and its predictive value for the recurrence after transurethral resection of bladder tumor.A retrospective analysis was made of 194 specimens who underwent transurethral resection of bladder tumors in our hospital from January 2014 to January 2016 and completed follow-up. The expression intensity of CA-IX and the clinical data of the patients were analyzed, and the subjects were divided into positive group and negative group according to the expression intensity of CA-IX. The age, gender, T stage, degree of differentiation, tumor number, tumor diameter, recurrence of each group was analyzed. Logistic univariate and multivariate analysis was used successively to find independent influencing factors for predicting the recurrence of bladder urothelial carcinoma after resection. The Kaplan–Meier survival curve was drawn according to the relationship between CA-IX expression intensity and postoperative recurrence.P > 0.05), while the positive expression of CA-IX had statistical significance with tumor T stage, tumor differentiation, tumor number and recurrence (P < 0.05); Logistic regression analysis showed that clinical T stage, tumor differentiation, tumor number, and CA-IX expression intensities were independent risk factors for predicting recurrence of bladder urothelial carcinoma after resection (P < 0.05); There were 59 cases of recurrence in the positive expression of CA-IX group, with a recurrence rate of 44.69% (59/132), and 17 cases of recurrence in the negative expression group, with a recurrence rate of 27.41% (17/62). The mean recurrence time of CA-IX positive group was 29.93 ± 9.86 (months), and the mean recurrence time of CA-IX negative group was 34.02 ± 12.44 (months). The Kaplan–Meier survival curve showed that the recurrence rate and recurrence time of patients with positive expression of CA-IX in bladder urothelial carcinomas were significantly higher than those of patients with negative expression of CA-IX.The positive expression rates of CA-IX in bladder urothelial carcinomas were 68.1% (132/194). The positive expression of CA-IX had no statistical significance with age, gender and tumor diameter (CA-IX is highly expressed in bladder urothelial carcinoma, is a good tumor marker, and can be used as a good indicator for predicting the recurrence of bladder urothelial carcinoma after transurethral resection of bladder tumor. Bladder urothelial carcinoma is a common disease worldwide, and its mortality rate ranks 13th . TherefoBladder cancer can be divided into non-muscle invasive bladder cancer (NMIBC) and muscle invasive bladder cancer (MIBC), Non-muscle invasive bladder cancer occurs in approximately 70 to 80 percent of patients with primary diagnosis . TransurIt CA-IX is a member of the CA family , which iInclusion criteria: From January 2014 to January 2016, the patients were diagnosed with non-muscle invasive bladder urothelial carcinoma in the Department of Pathology of the First People's Hospital of Huzhou City, Zhejiang Province, and underwent transurethral resection of bladder tumor (TURBT).Exclusion criteria: Patients with incomplete clinical data; Patients with lymph node or distant metastasis; Patients receiving preoperative radiotherapy, chemotherapy, immunotherapy or targeted therapy; Patients with other malignant tumors; Patients with severe cardiovascular, liver and kidney, and blood system diseases; Immunocompromised persons.To those patients who underwent transurethral resection of bladder tumor (TURBT), Intravesical instillation of Epirubicin (50 mg) was performed regularly within 1 year after the operation; B-ultrasonography and cystoscopy were performed every 3 months after the operation. Complete the 5-year follow-up plan.Histological grading using the WHO 2004 grading system; Pathological staging using the 2009 TNM staging system (UICC).The content of this study has been approved by the Ethics Committee of our hospital.Rabbit anti-human CA-IX polyclonal antibody, mouse anti-human Ki-67 monoclonal antibody, immunohistochemistry EnVision kit, and DAB chromogenic kit were purchased from Fuzhou Maixin Biotechnology Development Co., Ltd.Take the archived wax block, make 3 serial sections of 4 μm thickness with a tissue microtome, 1 section is stained with HE, and the other 2 sections are attached to the glass slides pretreated with L-polylysine. The oxidase blocking solution was incubated at room temperature, soaked and rinsed in PBS, then placed in 0.01 mol/L citrate buffer (pH 6.0) for microwave repair, and cooled to room temperature. Blocked with 10% normal goat serum, incubated at room temperature, and added CA-IX and Ki-67 primary antibodies (1:100) overnight at 4 °C. Add biotin-labeled secondary antibody dropwise, and then dropwise add horseradish peroxidase-labeled streptavidin. DAB staining, hematoxylin counterstaining, and neutral gum mounting. Known positive sections were used as positive controls and phosphate-buffered saline (PBS) was used instead of primary antibodies as negative controls. Positive and negative control groups were set in each experiment. The experimental steps were strictly in accordance with the reagent instructions.CA-IX is located in the cell membrane, and the corresponding position is light yellow to dark brown as the positive expression of the cell. Randomly select 10 high-power fields for each slice, count the total number of cells and the number of positive cells, and score according to the percentage of positive cells: < 10% is 0 points, 10%–50% is 1 point, 51%–75% is 2 points, 76%–100% is 3 points; according to the staining intensity score: 1 point for no staining, 2 points for light staining, 3 points for medium staining, and 4 points for dark staining. The product of the positive cell percentage score and the color intensity score is used as the judgment result: ≤ 3 is divided into no expression (-), 4 to 6 is divided into low expression, and 7 to 12 is divided into high expression. In the end, no expression or low expression is regarded as negative expression of CA-IX; high expression is regarded as positive expression of CA-IX. Two senior pathologists used a double-blind method to read the film.χ2 test was used. P < 0.05 was considered statistically significant. Divided 194 bladder cancer patients into positive and negative groups according to the expression intensity of CA-IX; the correlation between the expression intensity of CA-IX and various parameters was analyzed. Univariate Logistic regression analysis was performed to find the risk factors of recurrence of bladder urothelial carcinoma after TURBT, furthermore, the factors obtained from univariate Logistic regression analysis were included in multivariate Logistic regression analysis to find the independent risk factors to predict recurrence of bladder urothelial carcinoma after TURBT (P < 0.05).The Kaplan–Meier survival curve was drawn based on the expression intensity of CA-IX, and the postoperative recurrence of the two groups was compared.SPSS 21.0 statistical software was used for statistical processing, the research data obeyed a normal distribution, and the P < 0.05). See Table Immunohistochemistry showed that CA-IX was expressed on the cell membrane, The positive rate of CA-IX in bladder urothelial carcinoma was 68.1% (132/194), the positive rate in adjacent tissues was 44.3% (86/194), and the positive rate in normal tissues was 27.3% (53/194). The expression of CA-IX in bladder urothelial carcinoma was significantly higher than that in adjacent tissues and normal tissues ; the expression of CA-IX had statistical significance with T stage, degree of differentiation, tumor number and recurrence (P < 0.05). See Table The expression of CA-IX had no statistical significance with age, gender and tumor diameter  with T stage , degree of differentiation , CA-IX expression . Bladder urothelial carcinoma recurrence after TURBT had no statistical significance (P > 0.05) with age , gender , tumor number  and tumor diameter  (P > 0.05). , degree of differentiation ),the number of tumors  and CA-IX expression intensity  were independent influencing factors for predicting the recurrence of bladder urothelial carcinoma after resection (P < 0.05); Tumor diameter  was not statistically significant with recurrence after resection (P > 0.05). See Table The risk factors were further included in the multivariate Logistic regression analysis according to the univariate Logistic regression analysis. After analysis, we obtained: clinical T stage of bladder urothelial carcinoma . Among them, 59 cases relapsed in CA-IX positive expression group, the recurrence rate was 44.69% (59/132), and 17 cases relapsed in the negative expression group, and the recurrence rate was 27.41% (17/62). The mean recurrence time of CA-IX positive group was 29.93 ± 9.86 (months), and the mean recurrence time of CA-IX negative group was 34.02 ± 12.44 (months), P < 0.05). See Fig. According to the expression of CA-IX, the relapsed patients were divided two groups, and then the Kaplan–Meier survival curve was drawn. The curve showed that the recurrence rate of CA-IX positive expression group was significantly higher than that of negative group , and the positive rate in adjacent tissues was 44.3% (86/194), the positive rate of normal tissue was 27.3% (53/194). This revealed that CA-IX is significantly expressed in bladder urothelial carcinoma and is a meaningful tumor marker for bladder urothelial carcinoma, which may provide a new method for molecular diagnosis of bladder urothelial carcinoma .P = 0.01), the degree of differentiation : 0.262–0.956, P = 0.036) and CA-IX expression intensity  were independent influencing factors for predicting the recurrence of bladder urothelial carcinoma after resection. This indicates that CA-IX plays an important role in the occurrence and development of bladder urothelial carcinoma. This result also confirms the research of domestic and foreign scholars [The recurrence rate of bladder urothelial carcinoma after resection is high, and it is currently recognized that the degrees of differentiation, tumor number, tumor diameter, and pathological stage are risk factors for predicting the recurrence of bladder urothelial carcinoma , 25. In P < 0.05) by plotting the Kaplan–Meier survival curve. This phenomenon indicates that the expression of CA-IX is a meaningful tumor marker for predicting the recurrence of bladder urothelial carcinoma. Combined with the expression intensity of CA-IX before surgery, it can provide a reference for the selection of surgical methods and improve the therapeutic effect. Patients with positive CA-IX expression or a large number of tumors, poor differentiation, and advanced stages should be followed up closely after surgery to avoid missing recurrence or progression, which may lead to the progression of some patients to muscle-invasive bladder cancer, eventually having to undergo radical cystectomy. Our study shows that high CA-IX expression can lead to poor prognosis, positive expression is closely related to recurrence rate, CA-IX is an independent risk factor for predicting recurrence of bladder urothelial carcinoma after resection.We conducted long-term follow-up of surgical patients. 245 patients were enrolled, and a total of 194 patients completed the 5-year follow-up, 42 patients were lost to follow-up, 9 patients had incomplete data, and 194 patients finally completed the follow-up plan. During the follow-up period, 76 cases recurred, 118 cases did not recur, and the recurrence rate was 39.17% (76/194). Among them, 59 cases relapsed in CA-IX positive expression group, the recurrence rate was 44.69% (59/132), and 17 cases relapsed in the negative expression group, and the recurrence rate was 27.41% (17/62). The mean recurrence time of CA-IX positive group was 29.93 ± 9.86 (months), and the mean recurrence time of CA-IX negative group was 34.02 ± 12.44 (months). It was found that the recurrence rate and recurrence time of patients with positive expression of CA-IX in bladder urothelial carcinoma were significantly higher than those of patients with negative expression of CA-IX (To sum up, It CA-IX is an excellent tumor marker for bladder urothelial carcinoma, but it is not a specific tumor marker of bladder, and the expression of CA-IX can be used to predict bladder urothelial carcinoma after resection. There is a certain reference value later. It CA-IX protein has a certain role in the occurrence and progression of bladder urothelial carcinoma, which is a topic worthy of further study, and can provide a new direction for the development of targeted drugs. However, the sample of this study is limited, the experimental design is a retrospective study, and the postoperative follow-up period is not long, and the final conclusion needs further research to confirm, In addition, the expression and effect of CA-IX in bladder muscle-invasive urothelial carcinoma is also worth studying.


Objective: A sensitive and selective fluorescence-detection platform based on carbon quantum dots (CQDs) was designed and developed for the determination of methotrexate (MTX), for the purpose of minimizing the possible toxic threat of MTX in clinics. Methods: The approach was prepared for the first time by a simple, hydrothermal method, making the synthesis and modification processes realized in one step using polyethyleneimine (PEI), and the proposed PEI-CQDs were obtained with high fluorescence quantum yield (38%). Results: MTX was found highly responsive and effective in quenching the fluorescence of the PEI-CQDs, due to a suggested fluorescence resonance energy transfer mechanism or inner-filter effect. The linear range of MTX was between 1 and 600 μmol/L under optimum conditions, with a detection limit (LOD) as low as 0.33 μmol/L. Furthermore, the fluorescent method was established for the MTX assay, and satisfactory results were acquired in real-sample determination. The average labeled quantity was 98.2%, and the average added standard recovery was 100.9%. Conclusions: The proposed PEI-CQDs showed a remarkable potential for broad applications in biological molecule determination and environmental analysis. Currently, methotrexate (MTX) is a commonly used anti-folic acid and anti-tumor drug, mainly applicable to leukemia meningeal spinal infiltration, acute leukemia, head and neck tumors, bone tumors, breast cancer, liver cancer, lung cancer, and other diseases ,2. MTX hAt present, among various detection techniques applied to MTX, for example, are electrochemical ,10,11, lCarbon quantum dots (CQDs) are a new class of carbon nanomaterials. They are quasi-spherical carbon nanoparticles, with a core–shell structure and a size of less than 10 nm. They exhibit several remarkable properties, including ease of preparation, non-toxicity, and excellent luminescence behavior ,18,19. CPolyethylene imine (PEI) is a type of amphiphilic cationic polymer . Due to In conclusion, we propose a novel fluorescence probe based on PEI-CQDs for the fast and selective determination of MTX. The obtained fluorescence probes were prepared by a simple, hydrothermal method, making the synthesis and modification processes realized in one step. Moreover, the fluorescence quantum yield (QY) of the PEI-CQDs was 38%, and the PEI-CDs were used as the fluorescent probes for the determination of MTX in the phosphate buffer solution (PBS) and even in human serum, exhibiting not only good sensitivity but also satisfactory practicability.−1. The surface and functional properties of PEI-CQDs were characterized using an X-ray diffractometer (XRD) equipped with a D8 advance . The photo-luminescence (PL) spectra of the samples were recorded on a UV-2501PC spectrophotometer  and an F-7000 fluorescence spectrophotometer , respectively. Transmission electron microscopy (TEM)  with a 200 kV operating voltage was used to characterize the microstructure and the sizes of the PEI-CQDs. Fourier-transform infrared (FTIR) spectra of pressed KBr pellets were recorded using an AVATAR-370 spectrometer  over the spectral range 400–4000 cmMethotrexate hydrate (>97%) was obtained from Shanghai Titan Technology Co., Ltd. . Polyethyleneimine  was purchased from Shanghai Vokai Chemical Reagent Co., Ltd. . Sinopharm Chemical Reagent Co., Ltd.  provided the ethanediol (>99.5%) and the remaining reagents used in this work. Shanghai Xinyi Pharmaceutical Co., Ltd.  produced the methotrexate tablets. All analytical measurements were performed using ultrapure water (18.25 MΩ cm).g rpm to remove large carbon particles and unreacted substances, and the upper solution was filtered with a 0.22 µm filter membrane, with the constant volume at 50 mL, and then stored away from light for later use.PEI-CQDs were synthesized by a hydrothermal technique that utilized glycol as a source of carbon and PEI as a passivating agent for the surface. This approach was previously described in another study, with slight modifications . First, QY) of PEI-CQDs can be determined and calculated by the following equation [RQY = 54%. I and RI are the measured, integrated emission intensity of PEI-CQDs and quinine sulfate, respectively. A and RA represent the absorbance of PEI-CQDs and quinine sulfate, which are measured on a UV–Vis spectrophotometer. The quinine sulfate was dissolved in 0.1 M of H2SO4 (n = 1.428), and the PEI–CQDs were dissolved in distilled water (n = 1.333).The fluorescence quantum yield (equation .(1)QY=QA total of 10 MTX tablets were weighed and fully ground. Then, the powder was extracted with 0.1 mol/L of NaOH solution for 30 min in an ultrasonic bath until sufficiently dissolved, transferred to a clean, 100 mL volumetric flask for constant volume, and mixed well. The solution was diluted to the exact volume with distilled water, and then filtered by a 0.22 μm filter membrane. At the end, the sample solution was 1 mg/mL, and it was stored in the dark and was used as the real sample’s solution.The following procedure was used to determine the presence of MTX in real samples: 250 μL of PEI-CQDs solution was poured into a series of colorimetric tubes (10 mL), and then 0.01 mol/L of MTX solution in different volumes was added to each colorimetric tube in turn, followed by the phosphate buffer solution (PBS) (pH 7.0) as the calibration line. After 3 min, the fluorescence intensity was detected at an excitation wavelength of 350 nm.During the synthesis of PEI-CQDs, the amine group in dendritic PEI can react with the hydroxyl group in glycol to form a hydrogen bond and coat the carbon point. The carbon elements on the surface of the fluorescent carbon spots form carboxyl groups under the oxidation of concentrated phosphoric acid, making them hydrophilic and providing active groups for subsequent biological applications to improve luminescence performance. The QY of PEI-CQDs was calculated using the method described, and the result was 38%, according to −1 is attributed to N-H and O-H stretching vibrations, and the absorption peak is stronger than those of ethylene glycol and PEI, showing the presence of amino and hydroxyl groups on the surface of the PEI-CQDs, which contributes to their hydrophilic properties. The peaks at 1653 cm−1 and 1380 cm−1 corresponded to the NH/NH2 N-H stretching vibration and the C-H stretching vibration, respectively. The absorption peak at 1095 cm−1 is attributed to the stretching vibration of C-O-C [The chemical composition and functional groups on the surface of the developed PEI-CQDs were investigated. The FT-IR spectrum of the synthesized PEI-CQDs is shown in of C-O-C .n−π * transition, while PEI have nearly no absorption above 240 nm, which was a difference between PEI-CQDs and PEI [UV-vis absorption and photoluminescence-emission spectroscopy were used to characterize the optical properties of PEI-CQDs. As illustrated in  and PEI . Meanwhi0, and F was the fluorescence intensity when MTX was added. In 0 − F) was obtained.Several related factors, such as the pH and the volume of the PEI-CQDs solution, were investigated. As illustrated in To assess the analytical performance, MTX samples at various concentrations were mixed with PEI-CQDs under optimal conditions. The fluorescence quenching of PEI-CQDs toward MTX appeared at concentrations ranging from 1 to 600 μmol/L, as shown in 0/F) = 0.00284 CMTX + 0.04575, which has a correlation coefficient of R2 = 0.9976. At a signal/noise ratio of 3, the limit of detection (LOD) is 0.33 μmol/L and the limit of quantitation (LOQ) is 1.0 μmol/L. This fluorescent method, as developed, can successfully be used to monitor MTX in real time. The presented method can provide a wide linear range with other previous methods , and the results are shown in The developed method’s applicability was evaluated using MTX tablets purchased from a local drug store. The standard addition method was performed in human serum provided by different healthy volunteers from the infirmary of Shanghai University. The 100 μL human serum was diluted 100 times with PBS (pH 7.0), and then 100 μL supernatant containing MTX (1 mg/mL) was added. The average labeled quantity was 98.2%, with a relative standard deviation (RSD) of 4.4% . FurtherIn conclusion, PEI as a surface passivation agent that modified CQDs was successfully prepared using a one-step hydrothermal method with excellent water solubility and higher quantum yield. The conditions were optimized. The fluorescence quenching of PEI-CQDs by MTX was caused primarily by an inner-filter effect based on the agglomeration and electron transfer of PEI-CQDs. The as-prepared PEI-CQDs demonstrated excellent MTX-detection performance, with linear dynamic ranges ranging from 1 to 600 μmol/L and a detection limit of 0.33 μmol/L. Furthermore, the MTX spiked in drug samples monitored by the method recovered well. We anticipate that this approach can be used for developing more sensitive sensing methods in drug analysis and environmental monitoring.


Background: The rapid worldwide spread of the Omicron variant of SARS-CoV-2 has unleashed a new wave of COVID-19 outbreaks. The efficacy of molnupiravir, an approved drug, is still unknown in patients infected with the Omicron variant.Objective: Evaluated the antiviral efficacy and safety of molnupiravir in patients infected with SARS-CoV-2 Omicron variant, with symptom duration within 5 days.Methods: We conducted a randomized, controlled trial involving patients with mild or moderate COVID-19. Patients were randomized to orally receive molnupiravir (800 mg) plus basic treatment or only basic treatment for 5 days (BID). The antiviral efficacy of the drug was evaluated using reverse transcriptase polymerase chain reaction.Results: Results showed that the time of viral RNA clearance (primary endpoint) was significantly decreased in the molnupiravir group  compared to the control group  (Log-Rank p = 0.0092). Of patients receiving molnupiravir, 18.42% achieved viral RNA clearance on day 5 of treatment, compared to the control group (0%) (p = 0.0092). On day 7, 40.79%, and 6.45% of patients in the molnupiravir and control groups, respectively, achieved viral RNA clearance (p = 0.0004). In addition, molnupiravir has a good safety profile, and no serious adverse events were reported.Conclusion: Molnupiravir significantly accelerated the SARS-CoV-2 Omicron RNA clearance in patients with COVID-19.Clinical Trial Registration: [chictr.org.cn], identifier [ChiCTR2200056817]. The Omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has rapidly replaced the Delta variant as the dominating SARS-CoV-2 variant, which has higher infectivity and stronger vaccine breakthrough ability . Among tin vitro and in vivo . The protocol was reviewed and approved by the Medical Ethics Committee of the Third People’s Hospital of Shenzhen, and the trial was conducted in accordance with the Declaration of Helsinki and Good Clinical Practice guidelines of the International Conference on Harmonization. Molnupiravir was provided by HUAHAI Pharmaceutical.From March 3 to 21 March 2022, patients with confirmed mild/moderate COVID-19 were admitted and enrolled at the Third People’s Hospital of Shenzhen. Eligible patients include male or nonpregnant females aged ≥18 years and ≤80 years, who were tested positive for SARS-CoV-2 Omicron variant and had initial onset of symptoms for ≤5 days prior to the day of treatment. Exclusion criteria included severe vomiting or intolerance of oral drugs for other reasons, pregnancy or lactation, patients retesting positive for COVID-19, and those who had received antibody therapy, plasma therapy, or other investigational drugs for SARS-CoV-2. All the patients provided a written informed consent.n = 80) and control groups (n = 36) using a random number table. Molnupiravir (800 mg twice per day) were administered orally for 5 days in the hospital. In addition, all patients received the same basic treatment, which consisted of vitamin C, lianhuaqingwen granule, and nasal irrigation. Pharyngeal swab was collected every other day to determine the antiviral effect of molnupiravir against SARS-CoV-2 using RT-PCR. Adverse events were monitored throughout the study period.Patients were randomly grouped (2:1) into molnupiravir . Samples were tested for detection of ORF1ab and N genes. A negative RT-PCR result was confirmed only when all test results were reported negative (Ct > 35). The secondary antiviral efficacy endpoint included the percentage of patients who were negative for SARS-CoV-2 infectious virus on days 5, 7, and 10.p value <0.05 was considered statistically significant. Time of viral RNA clearance was summarized using the Kaplan-Meier methodology, and log-rank test was used for comparison of the efficacy between treatments. The percentage of patients who were negative for viral RNA was summarized and compared between groups using Fisher’s exact test.The measurement data conforming to non-normal distribution were exhibited as median, and the Wilcoxon rank-sum test was used for comparison between groups. Meanwhile, the count data were exhibited as cases (%), and comparisons between groups were made using the chi-square test. A From March 3 to 21 March 2022, 116 patients were randomly grouped, with 108 receiving molnupiravir or only basic treatment . The patp = 0.0092, A total of 107 patients in the cohort were included for the primary efficacy analysis. One patient in the molnupiravir group was excluded from the antiviral efficacy analysis because of a negative result in the viral RNA test less than 12 h after taking the medicine. The primary outcome of this study was the time of viral RNA clearance. The median time of viral clearance were 9 days (95% CI: 7–9 days) and 10 days (95% CI: 9–11 days) in the molnupiravir and control groups, respectively. The median time was significantly reduced in the molnupiravir group (log-rank p = 0.0092). The percentage of patients who were negative for viral RNA also increased to 40.79% on day 7 in the molnupiravir group compared to the control group (6.45%) (p = 0.0004). The percentage of patients negative for viral RNA remained significantly different between molnupiravir and control groups on day 10 of treatment  (p = 0.02).The key secondary virological endpoint of this trial was the percentage of patients who were negative for viral RNA on days 5, 7, and 10 . On day Three adverse events were reported in the molnupiravir group . One patp = 0.499), and the results were consistent with previously published studies (p = 0.096).The duration of fever, time of symptom alleviation and laboratory test results were collected to evaluate the overall health condition associated with COVID-19. At baseline, patients in the molnupiravir and control groups matched well in all presentation indicators, with no significant difference. In the laboratory test before discharge, all test values of patients recovered to the normal range, and no significant difference was observed between the groups . The med studies . In addiAs of 22 March 2022, 470,839,745 confirmed COVID-19 cases were recorded, including 6,092,933 deaths, and a total of 10,925,055,390 doses of vaccine had been administered worldwide. The emerging Omicron variant of SARS-CoV-2 is highly infectious and has a stronger vaccine breakthrough ability against antibodies provided by existing vaccines, thus it is the dominating SARS-CoV-2 variant globally . The curp = 0.013) (p = 0.0092). This difference may be due to two possible reasons, as follows: 1) reduced replication capacity and pathogenicity of the Omicron variant compared to the other variants (p = 0.521)  . In thisvariants , and 2) variants . We have= 0.521) . In termBased on the higher infectivity and reduced pathogenicity of the Omicron variant, it is more likely to result in a great deal of patients with mild COVID-19 or asymptomatic infections, rather than severe COVID-19. As a result, the clearance of viral RNA from patients and interruption of viral transmission are more relevant in the fight against Omicron variant infections. This clinical trial was outlined to assess the efficacy and safety of molnupiravir against the Omicron variant of SARS-CoV-2 and did not focus on evaluating clinical endpoints, such as symptom duration or length of hospitalization. Furthermore, recruitment was not limited to patients with risk factors of progressing to severe disease. Patients did not benefit from molnupiravir treatment in terms of duration of fever and symptoms alleviating time during the observation period. Another limitation is that asymptomatic individuals were not recruited into the trial. In this population, which was positive for viral RNA but has no characteristic symptoms, treatment with molnupiravir that exert a direct antiviral effect is equally relevant and may be more effective.This study provides biological evidence to support the use of molnupiravir in accelerating viral RNA clearance in patients infected with the Omicron variant of SARS-CoV-2, potentially reducing viruses early transmission. The dynamic zero COVID-19 strategy is the most effective prevention and control strategy proposed and implemented in China during the COVID-19 epidemic. In the face of a new wave of COVID-19 outbreaks caused by the Omicron variant, small-molecule oral drug therapy will contribute to the implementation of the dynamic zero COVID-19 strategy and accelerate the end of the epidemic.